Role of the TOC complex in plastid protein import

描述（由申请人提供）：细胞器的识别和发育依赖于一套复杂的细胞内蛋白质运输系统，该系统介导核编码蛋白特异性靶向到其适当的亚细胞区室。这些贩运系统不仅必须保持高度的特异性，而且必须适应发展事件所造成的贩运基质水平和组成的巨大变化。贩运系统的缺陷导致许多人类疾病，包括齐薇格综合征（过氧化物酶体生物发生）和耳聋/肌障碍综合征（线粒体蛋白输入），或具有阻止细胞生长和发育的致命后果。植物叶绿体中蛋白质输入的研究是研究真核细胞中蛋白质运输和细胞器生物发生的有力模型，因为它允许在多细胞生物中进行生物化学和遗传分析。本研究的长期目标是明确导入过程的分子机制，了解导入在质体发育中的作用。本文主要研究叶绿体外包膜转运子（Toc复合体）的两个gtpase Toc34和Toc159在蛋白前体细胞识别和膜转运调控中的作用。我们将验证Toc159作为初始前蛋白受体的假设，以及gtp调节的与Toc34的异源二聚化有助于调节前蛋白向膜易位通道的转移，从而使前蛋白进入进口途径。为此，我们将利用拟南芥Toc159 （at Toc159）和Toc34 （atToc33）的零突变体来建立Toc GTPase功能的层次结构，并研究每种蛋白在导入反应中的具体作用。此外，将采用生物化学和遗传学相结合的方法来解决Toc组分的GTPase活性在调节前蛋白识别和进入易位通道中的作用。最终目的是验证这样一个假设，即Toc159和Toc34家族的不同成员代表了蛋白质靶向质体的不同途径，并且不同的途径已经进化以确保质体发育过程中必需蛋白质的平衡进口。将定义不同跨区域形成和功能的结构基础。这些信息，结合Toc159四个基因突变体的组织特异性缺陷信息，将有助于深入了解细胞器和组织发育过程中不同蛋白质输入途径的作用。

DESCRIPTION (provided by applicant): Organelle identity and development rely on a complex set of intracellular protein trafficking systems that mediate the specific targeting of nuclear-encoded proteins to their proper subcellular compartment. Not only must these trafficking systems maintain a high degree of specificity, they must adapt to accommodate dramatic changes in the levels and composition of trafficking substrates imposed by developmental events. Defects in trafficking systems lead to numerous human disorders, including Zellweger syndrome (peroxisome biogenesis) and deafness/dystonia syndrome (mitochondrial protein import), or have lethal consequences that prevent cell growth and development. The study of protein import into plant chloroplasts is a powerful model for investigating protein trafficking and organelle biogenesis in eukaryotic cells because it allows combined biochemical and genetic analyses in a multicellular organism. The long-term goal of this research is to define the molecular mechanism of the import process and understand the role of import in plastid development. This proposal focuses on the role of two GTPases of the translocon at the outer envelope membrane of chloroplasts (Toc complex), Toc34 and Toc159, in preprotein recognition and the regulation of membrane translocation. We will test the hypothesis that Toc159 serves as the initial preprotein receptor and that GTP-regulated heterodimerization with Toc34 serves to regulate transfer of preproteins into the membrane translocation channel, thereby committing preproteins to the import pathway. To this end, we will take advantage of null mutants of Arabidopsis Toc159 (at Toc159) and Toc34 (atToc33) to establish the hierarchy of Toc GTPase function and examine the specific roles of each protein in the import reaction. In addition, combined biochemical and genetic approaches will be used to address the role of the GTPase activities of the Toc components in regulating preprotein recognition and access to the translocon channel. The final aim will test the hypothesis that the different members of the Toc159 and Toc34 families represent distinct pathways for protein targeting to plastids, and that separate pathways have evolved to ensure balanced import of essential proteins during plastid development. The structural basis for the formation and function of distinct translocons will be defined. This information, combined with information on the tissue-specific defects of mutants in each of the four at Toc159 genes, will provide insight into the roles of distinct protein import pathways during organelle and tissue development.